Sidsel Markussen

Interactions of the bacteriocins sakacin P and nisin with food constituents

Bacteriocins are amphiphilic peptides susceptible to adsorption to food macromolecules and proteolytic degradation. These properties may limit their use as preservation agents. The aim of the present work has been to elucidate the fate of the bacteriocin sakacin P in food. Nisin was used in a few experiments for comparison. Recovery of bacteriocins was studied in homogenates of cold-smoked salmon, chicken cold cuts and raw chicken, with verification of the results in the corresponding food products. More than 80% of the added sakacin P and nisin were quickly adsorbed to proteins in the food matrix. In foods that had not been heat-treated, proteolytic activity caused a rapid degradation of the bacteriocins, with less than 1% of the total activity left after 1 week in cold-smoked salmon, and even less in raw chicken. In heat-treated foods, the bacteriocin activity was stable for more than 4 weeks. The high fat content in salmon compared to chicken had no adverse effect on bacteriocin recovery or activity. However, mixing of triglyceride oils and bacteriocin solutions caused a considerable loss of activity. No principal differences were observed between sakacin P and nisin, but less nisin was adsorbed to muscle proteins at low pH, and the negative effect of oils was less pronounced for nisin. Growth of Listeria monocytogenes was completely inhibited for at least 3 weeks in both chicken cold cuts and cold-smoked salmon by addition of sakacin P (3.5 microg/g), despite the proteolytic degradation in the salmon.

Utilization of n-alkanes by a newly isolated strain of Acinetobacter venetianus: the role of two AlkB-type alkane hydroxylases

A bacterial strain capable of utilizing n-alkanes with chain lengths ranging from decane (C10H22) to tetracontane (C40H82) as a sole carbon source was isolated using a system for screening microorganisms able to grow on paraffin (mixed long-chain n-alkanes). The isolate, identified according to its 16S rRNA sequence as Acinetobacter venetianus, was designated A. venetianus 6A2. Two DNA fragments encoding parts of AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, were polymerase chain reaction-amplified from the genome of A. venetianus 6A2. To study the roles of these two alkM paralogues in n-alkane utilization in A. venetianus 6A2, we constructed alkMa, alkMb, and alkMa/alkMb disruption mutants. Studies on the growth patterns of the disruption mutants using n-alkanes with different chain lengths as sole carbon source demonstrated central roles for the alkMa and alkMb genes in utilization of C10 to C18 n-alkanes. Comparative analysis of these patterns also suggested different substrate preferences for AlkMa and AlkMb in n-alkane utilization. Because both single and double mutants were able to grow on n-alkanes with chain lengths of C20 and longer, we concluded that yet another enzyme(s) for the utilization of these n-alkanes must exist in A. venetianus 6A2.

Carbohydrate degradation and methane production during fermentation ofLaminaria saccharina (Laminariales, Phaeophyceae)

Anaerobic digestion of the brown algaLaminaria saccharina (L.) Lamour. harvested in spring and autumn was carried out at controlled laboratory conditions in stirred fermentor systems. Due to the normal seasonal variations, the autumn material had a much higher content of carbohydrates such as mannitol and laminaran. Both batch and semi-continuous feeding conditions were investigated for periods up to 800 h, with inoculum provided from previous kelp fermentations. In batch cultures, the methane yield from the autumn material was doubled compared to that of the spring material. Semi-continuous conditions gave more similar methane yields for both raw materials, 0.22 and 0.27 l CH4 per g VS for spring and autumn material, respectively. In all experiments, mannitol and laminaran were reduced to less than 5‰ of the initial values within 24–48 hours after inoculation, whereas 30‰ of the alginate content was detectable even after 30 days. Viscometry revealed that this material was severely depolymerized, and alginate lyase activity was found to develop rapidly in all cultures. Although mannitol and laminaran were fermented much faster than alginate, the total accumulated methane yields seemed to be determined by the total carbohydrate content of the raw material during extended semi-continuous feeding.

High coverage sequencing of DNA from microorganisms living in an oil reservoir 2.5 kilometres subsurface

Microorganisms colonize a variety of extreme environments, and based on cultivation studies and analyses of PCR-amplified 16S rDNA sequences, microbial life appears to extend deep into the earth crust. However, none of these studies involved comprehensive characterizations of total DNA. Here we report results of a high-coverage DNA pyrosequencing of an apparently representative and uncontaminated sample from a deep sea oil reservoir located 2.5 km subsurface, attributing a pressure and temperature of 250 bars and 85°C respectively. Bioinformatic analyses of the DNA sequences indicate that the reservoir harbours a rich microbial community dominated by a smaller number of taxa. Comparison of the metagenome with sequences in databases indicated that there may have been contact between the oil reservoir and surface communities late in the sequence of geological events leading to oil reservoir formation. One specific gene, encoding a putative enolase, was synthesized and expressed in Escherichia coli. Enolase activity was confirmed and was found to be much more thermotolerant than for a corresponding E. coli enzyme, consistent with the conditions in the oil reservoir.

Comparison of Different Strategies for Selection/Adaptation of Mixed Microbial Cultures Able to Ferment Crude Glycerol Derived from Second-Generation Biodiesel

Objective of this study was the selection and adaptation of mixed microbial cultures (MMCs), able to ferment crude glycerol generated from animal fat-based biodiesel and produce building-blocks and green chemicals. Various adaptation strategies have been investigated for the enrichment of suitable and stable MMC, trying to overcome inhibition problems and enhance substrate degradation efficiency, as well as generation of soluble fermentation products. Repeated transfers in small batches and fed-batch conditions have been applied, comparing the use of different inoculum, growth media, and Kinetic Control. The adaptation of activated sludge inoculum was performed successfully and continued unhindered for several months. The best results showed a substrate degradation efficiency of almost 100% (about 10 g/L glycerol in 21 h) and different dominant metabolic products were obtained, depending on the selection strategy (mainly 1,3-propanediol, ethanol, or butyrate). On the other hand, anaerobic sludge exhibited inactivation after a few transfers. To circumvent this problem, fed-batch mode was used as an alternative adaptation strategy, which led to effective substrate degradation and high 1,3-propanediol and butyrate production. Changes in microbial composition were monitored by means of Next Generation Sequencing, revealing a dominance of glycerol consuming species, such as Clostridium, Klebsiella, and Escherichia.

Time-resolved 1H and 13C NMR spectroscopy for detailed analyses of the Azotobacter vinelandii mannuronan C-5 epimerase reaction.

AlgE2, AlgE4, and AlgE6 are members of a family of mannuronan C-5 epimerases encoded by Azotobacter vinelandii, and are active in the biosynthesis of alginate, where they catalyze the post-polymerization conversion of beta-D-mannuronic acid residues into alpha-L-guluronic acid residues. To study the kinetics and mode of action of these enzymes, homopolymeric mannuronan and other alginate samples with various composition were epimerized by letting the enzymatic reaction take place in an NMR tube. Series of 1H NMR spectra were recorded to obtain a time-resolved picture of the epimerization progress and the formation of specific monomer sequences. Starting from mannuronan, guluronic acid contents of up to 82% were introduced by the enzymes, and the product specificity, substrate selectivity, and reaction rates have been investigated. To obtain direct information of the GulA-block formation, similar experiments were performed using a 13C-1-enriched mannuronan as substrate. The NMR results were found to be in good agreement with data obtained by a radioisotope assay based on 3H-5-labeled substrates.

Microbial communities of a complex high-temperature offshore petroleum reservoir

In this paper, the microbial communities of a complex high-temperature (appr. 85°) offshore petroleum reservoir are described as part of improving the understanding of the biogeochemical processes in the reservoir. Based on molecular biology studies, the results revealed that the microbial communities differed between samples from producing wells and the oil/water production facility of the reservoir, although a few types of thermophilic microbes appeared in both environments. The microbial communities of the different producing wells could be separated into three groups, and the target bacteria related to the genera Halomonas, Arcobacter or Pseudomonas seemed to be associated with these groups. Thus, different microbial communities were detected in this reservoir, and further investigations will clarify if specific target microbes may be used for the identification of different oil qualities or other reservoir characteristics. [Received: November 4, 2007; Accepted: January 10, 2008]

Influence of environmental conditions on the activity of the recombinant mannuronan C-5-epimerase AlgE2

The mannuronan C-5-epimerase AlgE2 is one of a family of Ca(2+)-dependent epimerases secreted by Azotobacter vinelandii. These enzymes catalyze the conversion of beta-D-mannuronic acid residues (M) to alpha-L-guluronic acid residues (G) in alginate. AlgE2 had a pH optimum between 6.5 and 7 and a temperature optimum around 55 degrees C. Addition of low molecular weight organic compounds, including buffers, amino acids and osmoprotective compounds, affected the activity of the enzyme. The charge, size and stereochemistry of the added compounds were important. The activity of AlgE2, dissolved in various buffers (same pH), decreased with increasing fraction of positively charged buffer ions. Mono- and divalent metal ions also influenced the activity. When Ca(2+) was omitted only Sr(2+), of the metal ions tested, supported some activity of AlgE2. At high concentration of Ca(2+) (3.3 mM) these ions had a negative effect on the activity, whereas at low Ca(2+) concentration (0.58 mM) the activity was enhanced by addition of Sr(2+), and to some degree also by addition of Mg(2+) and Mn(2+). During epimerization AlgE2 occasionally causes cleavage of the alginate chain. These chain breaks could not be prevented by changes in the conditions during the epimerization. The composition and sequential structure of epimerized alginate was not altered by changes in the epimerization conditions.

Chapter 1 Use of petroleum biotechnology throughout the value chain of an oil company: An integrated approach

Publisher Summary It is well established that petroleum reservoirs contain active and diverse populations of microorganisms. Microbial growth within oil reservoirs has been traditionally associated with biofouling and souring. The potentials for microbial improved oil recovery (MIOR) have been investigated for many decades. Nitrate injection was introduced as a method for curing reservoirs contaminated by sulphate-reducing prokaryotes. This chapter discusses petroleum biotechnology that possesses several other opportunities besides MIOR and nitrate injection. The primary target of the petroleum industry is to enhance and maintain a continuous oil production. An R&D program was initiated looking into the possibility of using petroleum biotechnology as an integrated approach throughout the value chain of the oil company. Its main objective was to evaluate biotechnology as a general toolbox for solving some of the technology problems of today, investigate future possibilities, and generate a resource base for new genetic information achieved from the organisms in the reservoir.

Valorization of Lignocellulosic Waste (Crotalaria juncea) Using Alkaline Peroxide Pretreatment under Different Process Conditions: An Optimization Study on Separation of Lignin, Cellulose, and Hemicellulose

ABSTRACT Lignocellulose materials, essentially consisting of lignin, cellulose and hemicelluloses, are abundant sources of fermentable sugars. The bast fiber of Crotalaria juncea (Sunn hemp), a native cover crop of India, was used as feedstock for this study. The primary objective of this study was to add value to the waste C. juncea bast fiber. The same was achieved by pretreating the waste fiber using alkaline peroxide solution at various process conditions. The optimal process condition for alkaline peroxide pretreatment was identified for the fiber to pretreatment solution (S/S) ratio of 1/40 at 50°C for 5h with respect to all response variables (lignin removal, hemicellulose recovery, recovery of solid pretreated material, and crystallinity of lignocellulose). Three-way ANOVA results revealed that S/S ratio had no significant effect; whereas, both temperature and time, and the combination of parameters, exhibited significant effect on response variables. The characteristic peaks associated with lignin and cellulose demonstrated a higher amount of lignin removal and increased cellulose content with elevated treatment time. Autoclave assisted pretreatment proved to be inefficient due to removal of lower amount of lignin in addition to higher hemicellulose degradation. On the other hand, pretreatment using ultrasound was found to be most effective in removing lignin, liberating hemicelluloses along with diminition in cellulose crystallinity.